N-acetylcysteine compositions and methods

ABSTRACT

Storage stable sterile ready-to-administer formulations comprising N-acetylcysteine are presented with desirable stability characteristics. In preferred aspects, formulations comprise low quantities of one or more chelating agents, contain N-acetylcysteine at a concentration of 25 mg/mL or 50 mg/mL, and are packaged in a suitable format, such as a polymeric bag with a metalized overwrap and a non-contact oxygen scavenger.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Non-Provisional PatentApplication with the Ser. No. 17/177,797, which was filed Feb. 17, 2021,which claims priority to U.S. Provisional Patent Application with theSer. No. 62/978,128, which was filed Feb. 18, 2020, the contents ofwhich are expressly incorporated by reference herein.

FIELD OF THE INVENTION

The present disclosure relates to pharmaceutical compositions andmethods, especially as they relate to storage stable andready-to-administer formulations of N-acetylcysteine.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Acetaminophen, commonly known as paracetamol, is a household analgesicmedication used to alleviate pain and fever. Acetaminophen istransformed in the liver to sulfate and glucuronide conjugates as formsof non-toxic metabolites that are then excreted in urine. However, asmall portion of acetaminophen is also oxidatively transformed byisozyme CYP2E1 of the cytochrome P450 mixed function oxidase enzymesystem in the liver to yield a reactive, and potentially toxic,intermediate called N-acetyl-p-benzoquinoneimine (NAPQI) as isschematically shown in FIG. 1 . This highly oxidative metabolite is astrong electrophile and is detoxified by glutathione (reduced form).Upon acetaminophen overdose, normal hepatic metabolism via sulfate andglucuronide cascades is overloaded, resulting in elevated NAPQI levels,which then deplete cellular glutathione storages. Consequently, excessNAPQI interacts with a variety of cellular components, and especiallyenzymes resulting in the disruption of function and overall cell death.Overdose by ingestion of 10-15 g of acetaminophen in a single dose wasreported to result in hepatic damage after 2-5 days with significantmorbidity resulting from complete hepatic failure.

Most commonly, acetylcysteine is administered intravenously for thetreatment of acetaminophen overdose. It is most effective in preventinghepatic toxicity if administered within 8-10 hours post acetaminophenoverdose. Acetylcysteine protects the liver by promoting glutathionesynthesis to replenish cellular stores. Deacetylation of acetylcysteinein the body yields L-cysteine that is consequently used to constructcellular glutathione. Moreover, acetylcysteine and L-cysteine can act asan alternative substrate to glutathione in detoxifying NAPQI. TheD-isomer of acetylcysteine has been reported to not preventhepatotoxicity unlike the L-isomer.

A half-life (t_(1/2)) of 5.7 hours was reported following an intravenousadministration of acetylcysteine. It has a complicated metabolic profileas it is transformed into several products (such as cysteine, cystine,methionine, glutathione, mixed disulfides, etc.) in the body. However,20-30% of administered acetylcysteine was reported to be excreted in theurine unchanged. Potential adverse reactions following intravenousadministration of acetylcysteine include acute flushing and erythema ofthe skin, and anaphylactoid reactions (now termed as nonimmunologicanaphylaxis) such as rash, urticaria, pruritis, hypotension, wheezingand/or shortness of breath.

Acetylcysteine, also known as N-acetyl-L-cysteine or N-acetylcysteine,is the acetyl derivative of the naturally occurring amino acidL-cysteine. Acetylcysteine is used in the treatment ofacetaminophen-overdose-induced hepatotoxicity. Acetylation of theN-terminus render the reactive amine into a relatively less reactiveamide. Acetylcysteine is a chiral molecule with the L-isomer beingactive while the D-isomer is inactive. Acetylcysteine exists as a whitecrystalline powder that has a melting point range of 109-110° C. andexudes a very slight acetic odor. Acetylcysteine is non-hygroscopic andis stable in ambient light.

Acetylcysteine is an antioxidant in biological systems and is aprecursor of the tripeptide glutathione that is responsible forneutralizing oxidative elements such as free radicals in humans andanimals. Acetylcysteine is deacetylated in biological systems (e.g.,blood, gut, etc.) to generate L-cysteine and therefore is considered aprodrug.

Solubility of acetylcysteine has been reported to be 1 gram in 5 mLwater or 4 mL alcohol. However, it is insoluble in chloroform and ether.It has three rotatable bonds, three hydrogen bond donors and threehydrogen bond acceptors. Acetylcysteine has an octanol/water partitioncoefficient (Log P) of −0.66 (estimated), reflecting its highly polarnature. Acetylcysteine does not react with glass, plastic, aluminum,anodized aluminum, chromed metal and stainless steel, however, solutionsof acetylcysteine react upon contact with rubber, some metals(particularly iron and copper surfaces), and/or when subjected toautoclaving, leading to discoloration and hydrogen sulfide generation.

Chemical stability issues, and particularly oxidative degradation inaqueous solutions of acetylcysteine are well known in the art, andvarious attempts have been made to solve problems associated withhydrolysis, nucleophilic attack, and oxidation. For example,acetylcysteine is stabilized in solution by inclusion of tromethamine asis described in CN 101239037. However, tromethamine may not be desirablein all uses of acetylcysteine. In further known methods, WO 2007/024311describes preparation of a concentrated solution of acetylcysteine whereoxygen depleted solvent at pH 6.8 provided unexpected chemical stabilityof the solution. However, such solution is a concentrate and requiresappropriate dilution prior to use. In yet other known methods,acetylcysteine is provided as a lyophilized powder along with a bufferagent as described in CN 101028252. Once more, reconstitution isrequired prior to administration, which adds delay to treatment andintroduces potential errors. Similarly, various concentratedacetylcysteine solutions are known in the art (e.g., ACETADOTE, 200mg/mL acetylcysteine concentrate by Cumberland Pharmaceuticals Inc.),which once more require dilution.

Regardless of the particular formulation, concentrated acetylcysteinesolutions present a further potential hazard as such concentratedsolutions require significant calculations to determine the appropriatedilution factor and volume for a given administration. In particular,acetylcysteine administration is not only dependent on the body weightof the subject receiving the medication, but also administered in threedistinct dosages: a higher loading dose (150 mg/kg), a lower second dose(50 mg/kg), and an intermediate third dose (100 mg/kg). As such,multiple administrations at different dosages introduce multiple pointsof potential contamination and dosing errors.

Thus, even though various acetylcysteine formulations are known in theart, all or almost all of them suffer from various disadvantages.Consequently, there is a need to provide improved compositions andmethods that provide storage stable and ready-to-administer formulationsof N-acetylcysteine that can reduce the risk of potential contaminationand dosing errors.

SUMMARY OF THE INVENTION

Compositions and methods are presented that provide desirable stabilityfor aqueous solutions of N-acetylcysteine, and especially forready-to-administer injectable formulations. Moreover, contemplatedcompositions will advantageously have a fixed concentration for use andwill so avoid potential risks due to contamination and/or miscalculationfor dilution. For example, contemplated formulations will compriseN-acetylcysteine at a fixed concentrations of about 25 mg/mL or 50 mg/mLand have a pH in the range of about pH 6.0-7.5 (e.g., pH 7.0),preferably in the presence of one or more metal chelators at lowconcentrations such as 10-60 mcg/mL.

In one aspect of the inventive subject matter, the inventors contemplatea method of administering N-acetylcysteine to a subject in need thereofthat includes a step of providing a sterile and ready-to-administercomposition comprising N-acetylcysteine containing N-acetylcysteine at afixed concentration, and a further step of administering, without priordilution, the ready-to-administer composition to a subject in needthereof in three distinct doses, wherein the fixed concentration of theN-acetylcysteine is the same in each of the three doses. Most typically,the ready-to-administer composition comprises a metal chelator (e.g.,ethylenediamine tetraacetic acid, preferably at a concentration ofbetween 10 and 60 mcg/mL) and has a pH between 6.0 and 7.5, andoptionally further includes an antioxidant and/or a tonicity agent(typically NaCl). The ready-to-administer composition presented hereinpreferably contains, after storage over at least three weeks at 80° C.,equal or less than 7%, equal or less than 6%, or equal or less than 5%total impurities generated from degradation of the N-acetylcysteine.

In some embodiments, the fixed concentration is 25 mg/mL or 50 mg/mL,and/or the ready-to-administer composition has a pH of 7.0±0.2. It isfurther contemplated that administration of a first dose and at leastpart of a second dose of the three doses may be done from a singlecontainer containing the ready-to-administer composition. Alternatively,administration of each of the three doses may also be done fromrespective separate containers, each containing the ready-to-administercomposition. Therefore, suitable containers may have a volume of between50 and 500 mL (e.g., between 200 and 300 mL). It is still furthercontemplated that the composition is filter sterilized or autoclaved.

In further embodiments, a first of the three doses is administered over1 hour, wherein a second of the three doses is administered over 4hours, and wherein a third of the three doses is administered over 16hours. Where desired, the administering the ready-to-administercomposition is performed using an infusion pump.

Therefore, the inventors also contemplate a sterile andready-to-administer N-acetylcysteine composition that includes anaqueous solution containing N-acetylcysteine at a concentration suitablefor direct administration via intravenous injection to a patient withoutdilution prior to administration. Most typically, such composition has apH of between 6.0 and 7.5 and comprises a chelating agent at aconcentration of between 10 and 60 mcg/mL. As noted before, it iscontemplated that the ready-to-administer composition contains, afterstorage over at least three weeks at 80° C., equal or less than 7% or 6%or 5% total impurities generated from degradation of theN-acetylcysteine. In preferred compositions the N-acetylcysteineconcentration is 25 mg/mL or 50 mg/mL, and the composition has a pH of7.0±0.2.

Viewed from another perspective, the inventors also contemplate a kitthat includes a primary container that contains a sterile andready-to-administer N-acetylcysteine composition as presented herein, asecondary container enclosing the primary container, and a non-contactoxygen absorbing agent in a space between the primary and secondarycontainer. Most typically, the primary container contains between 100and 500 mL of the ready-to-administer N-acetylcysteine composition, andthe secondary container is a polymeric container that may or may not beimpermeable to light or comprise a metal or metal layer.

In preferred aspects, the primary container contains 50 mL or 100 mL or150 mL or 200 mL or 250 mL or 300 mL or 350 mL or 400 mL or 450 mL or500 mL of the ready-to-administer N-acetylcysteine composition, and/orthe primary and secondary containers are polymeric bags. It is furthergenerally preferred that the ready-to-administer N-acetylcysteinecomposition is preservative-free.

Various objects, features, aspects, and advantages will become moreapparent from the following detailed description of preferredembodiments, along with the accompanying drawing in which like numeralsrepresent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically depicts exemplary degradation pathways foracetaminophen.

FIG. 2 schematically depicts exemplary cyclization reactions ofN-acetylcysteine at highly acidic conditions to form a thiazolinedegradation product and at highly basic conditions to for, athiazolidine degradation product.

DETAILED DESCRIPTION

The inventors have now discovered that sterile storage stable, aqueous,and ready-to-administer N-acetylcysteine formulations can be preparedthat not only have a desirable stability profile but that also have afixed concentration suitable for immediate administration to a subjectwithout need for prior dilution. Therefore, even complex modes ofadministration as discussed below can be performed in a very simplemanner that will entirely avoid potential risks associated with dilutionmistakes and microbial contamination.

Most preferably, contemplated compositions will contain acetylcysteineat a fixed concentration 25 mg/mL or 50 mg/ml and are packaged intosuitable containers such as IV bags at a volume of 200 ml or 300 ml bagfor an acetylcysteine concentration of 50 mg/ml or at a volume of 250 mlbag at an acetylcysteine concentration of 25 mg/ml. It should beappreciated that such presentation will not only avoid issues withon-site dilution, dilution errors, points of contamination, etc., butalso avoids osmolality/tonicity issues arising from different dilutionfactors needed for a concentrate (e.g., 200 mg/mL) to achieve a specificdosage regimen as is also discussed in more detail below. Viewed from adifferent perspective, contemplated compositions also eliminate helpreduce significant fluid loads to a subject. Still further, it should beappreciated that administration of specific dosages of the compositionspresented herein can be controlled via administered volume over timefrom a single container. Therefore, residual quantities of thecomposition can be ‘carried over’, for example, from a loading dose to amaintenance (2^(nd)) dose, or from a 2^(nd) dose to a 3^(rd) dose. Thus,and contrary to conventional practice, administration of a specific doseis not achieved by depletion of a fluid container that was prefilledwith a specific dose, but by typically metered delivery of apredetermined volume (e.g., via control of the drip rate forpredetermined time/volume). Moreover, the formulations presented hereincan be aseptically filled or terminally sterilized and packaged in aformat that maintains protection from oxygen ingress.

More specifically, and as is shown in more detail below, the inventorsdiscovered that formulations can be prepared that will contain afterstorage over at least three weeks less than 8%, or less than 7%, or lessthan 6%, or less than 5%, or less than 4%, or less than 3% of totalimpurities from the degradation of N-acetylcysteine as determined byHPLC-UV at 25° C. or even at accelerated storage conditions such asstorage at 40° C., or 60° C., or even 80° C. Notably, such stability atlow concentrations is particularly remarkable as these formulations willtypically contain very low concentrations of chelators, preferably lessthan 100 mcg/mL, or equal or less than 60 mcg/mL, or equal or less than50 mcg/mL, or equal or less than 40 mcg/mL, or equal or less than 30mcg/mL, or equal or less than 20 mcg/mL, or equal or less than 10mcg/mL. Thus, it is expected that the formulations presented herein willhave excellent storage stability of at least 1 month, or at least 2months, or at least 3 months, or at least 6 months, or at least 9months, or at least 12 months, or at least 18 months, or at least 24months, even at elevated storage temperatures (e.g., accelerated storageconditions such as 40° C., or 60° C., or even 80° C.). Moreover, suchformulations are also contemplated to be subjected to thermalsterilization, and particularly autoclaving to sterility (e.g., over atleast 5 min, or at least 10 min, or at least 15 min at 121° C.), withoutsubstantial increase in degradation.

Such stability is particularly remarkable in view of the diversepathways through which N-acetylcysteine can degrade. As is described inmore detail below, N-acetylcysteine can degrade though oxidation,hydrolysis, and nucleophilic reactions due to the presence of variousreactive functional moieties in N-acetylcysteine. For example,N-acetylcysteine can be subject to cyclization reactions under acidicand basic conditions to form 5-membered ring degradation products as isdiscussed in more detail below.

As noted above, acetylcysteine has various functional moieties thatrender the molecule susceptible to degradation under aqueous conditions.More specifically, acetylcysteine is very labile at extreme pHs and canundergo hydrolysis and nucleophilic reactions at highly acidic and basicpHs. Among other reactions, nucleophilic reactions can includeintramolecular cyclization and intermolecular reactions with excipients.Acetylcysteine is also highly susceptible to oxidizing agents such asreactive oxygen species and free radicals. Therefore, it should beappreciated that several degradative routes for acetylcysteine exist.

Hydrolysis: The amide bond in acetylcysteine imparts chemical labilityin the presence of nucleophiles at promoting conditions such as highlyacidic and basic pHs. Water, existing as predominantly hydroxyl orhydronium ions depending on the pH, can promote the hydrolysis of amidebond that resulting in the degradation of acetylcysteine to L-cysteineand acetic acid. The amide bond of amino acid-containing molecules suchas peptides and N-acetylated amino acids at neutral pH in the absence ofa catalyst (to lower bond breaking activation energy) is perceived to berobust. For instance, glycyl-D-valine amide/peptide bond was estimatedto have a half-life of 267 years at pH 7 and 37° C. However, the rate ofhydrolysis significantly increases once the pH moves towards theextremes (pH <5 and >9). Therefore, the inventors contemplate that a pHbetween the range of 5-9 may be suitable for a formulation ofacetylcysteine to minimize, if not prevent hydrolysis of the acetylamide bond.

Nucleophilic reaction: It has been very well documented that theelectron-rich thiol sidechain of Acetylcysteine act as a nucleophile atextreme pHs. At highly acidic pH, the carbonyl oxygen of the amide canabstract a proton in solution to exist as an activated cationic form.Intramolecular cyclization is initiated by nucleophilic attack ofthiol's sulfur atom resulting to a five-membered thiazolidine ring as isschematically shown in FIG. 2 . Subsequently, water is expelled togenerate a five-membered thiazoline ring in highly acidic solutions.While thiol sulfur can theoretically carry out a nucleophilic attack atthe carbonyl carbon of the carboxylic acid C-terminus (post protonationof the carbonyl oxygen at highly acidic pH to yield an activatedcationic form), cyclization resulting to four-membered rings arethermodynamically and kinetically unfavorable. Four-membered rings areinherently unstable due to bond constrains and therefore are notexpected to form under normal conditions. Conversely, the thiol groupcan be deprotonated at highly basic conditions promoting itsnucleophilic attack to the carbonyl carbon of the amide. Thisintramolecular reaction yields a five-membered thiazolidine ring as isschematically shown in FIG. 2 . In contrast to acidic media, loss ofwater cannot occur for a thiazolidine ring since basic conditionpromotes and stabilize the resulting anion, and that the generation of agood leaving group is prevented.

Intermolecular nucleophilic attack of acetylcysteine's thiol group alsooccurs at both acidic and basic pH. For instance, the thiol sulfur of amolecule can perform a nucleophilic attack on an activated carbonylcarbon of another molecule resulting to a dipeptide derivative. Thethiol group can also react with excipients having electrophilicmoieties. These can be particularly problematic under thermal conditionssince heat enhance the kinetics of reaction (introduced thermal energyincreases movements of particles and therefore their collision/reactionwith one another). Therefore, the inventors contemplate thatacetylcysteine should be maintained at neutral pH (5-9) to preventintramolecular cyclization reactions. Consequently, excipients withlabile electrophilic moieties such as reducing sugars are preferablylimited, if not entirely avoided in contemplated formulations.

Oxidation (redox reaction): Acetylcysteine readily loses electrons byone-electron or two-electron oxidation processes and reduce oxidizingagents such as free radicals. Depending on the oxidizing agent,different oxidation products and adducts are produced includingdisulfides such as cystine. Specifically, cystine is produced when thethiol group of two L-cysteine (deacetylated acetylcysteine) loseelectrons and react with one another forming a disulfide bond. Rateconstants for the reaction of acetylcysteine with various free radicalsand reactive compounds are known and exemplary data are shown inTable 1. Acetylcysteine is very labile in the presence of hydroxyl andnitrogen dioxide radicals. Hydroxyl radicals are typically generated insolution from peroxides such as hydrogen peroxide (H₂O₂) via divalentiron cation (Fe²⁺)-catalyzed Fenton reaction with molecular oxygen (O₂).Interestingly, Acetylcysteine is not reactive towards hydrogen peroxideas indicated by the rate constant of 0.85±0.09 M⁻¹s⁻¹. It has also beensuggested that molecular oxygen do not directly oxidize acetylcysteine.However, molecular oxygen indirectly affects the stability ofacetylcysteine as it participates in the formation of reactive oxygenspecies such as a hydroxyl radical and superoxide anion. Formation ofsuch free radicals and reactive oxygen species are promoted in thepresence of molecular oxygen and can be catalyzed by metal cations,thermal activation, and irradiation. The inventors therefore concludedthat the dissolved oxygen content should be minimized and that any metalcations in the formulation should be sequestered. It should be notedthat acetylcysteine can also chelate metal cations as described in moredetail below. In view of these considerations, the inventorsparticularly contemplate use of a metal chelator excipient that has ahigher metal complex formation constant (log K) for anticipated metalcations in the formulation as compared to the metal complex formationconstant of acetylcysteine.

TABLE 1 Reactive compound Rate constant (M⁻¹ s⁻¹) ExperimentalConditions Hydroxyl radical (^(•)OH)  1.36 × 10¹⁰  pH 7, RT Superoxideanion (O₂ ^(•−)) 68 ± 6 pH 7, RT Hydrogen peroxide (H₂O₂)  0.85 ± 0.09pH 7.4, 25° C. Peroxynitrite 415 ± 10 pH 7.4, 37° C. Nitrogen dioxideradical (^(•)NO₂) ~1.0 × 10⁷ pH 7.4, RT Nitroxyl (HNO)    5 × 10⁵ pH7.4, 37° C. NAPQI (1.36 ± 0.2) × 10⁴ pH 7, 25° C.

Peroxynitrite has been reported to react with acetylcysteine (at a rateconstant of 415±10 M⁻¹s⁻¹). It should be noted that peroxynitritereadily decomposes in solution to generate hydroxyl and nitrogen dioxideradicals and therefore much lability of acetylcysteine is due toperoxynitrite's degradants. Acetylcysteine is also reactive towardsnitroxyl (HNO) which is the reduced and protonated form of nitric oxide.These reactive nitrogen species can be present in nitrite-containingexcipients and/or from degradation thereof. Therefore, the inventorsalso contemplate that nitrites and nitrates should be avoided asexcipients or trace impurities.

Based on the above considerations, the inventors also investigatedadditional parameters, including redox potential, and metal chelationand exemplary considerations are as follows.

Redox Potential: Acetylcysteine was described to have a redox potentialof +63 mV relative to Glutathione (reduced GSH/oxidized GSSG) system asa reference standard. Thiol-containing molecules such as acetylcysteine,glutathione and L-cysteine are known to neutralize/reduce free radicals(oxidizing agents) by donating electrons from its electron-rich sulfuratom. Glutathione was described to be highly reducing at physiologicalconditions and have an experimental redox potential of −289 mV in thecytosol and therefore to an extent so does Acetylcysteine. Therefore,the inventors contemplate that it may be beneficial to omit the use ofhighly oxidizing agents in acetylcysteine formulations. Furthermore, theinventors contemplate that formation and dissemination of free radicalsin solution should be prevented to avoid oxidation of acetylcysteine.

Metal chelation: Acetylcysteine possess metal chelation capabilities dueto its thiol and carboxylic functional groups. Metal complexformation/stability constants (log K) for acetylcysteine at aqueoussolutions are shown in Table 2. Ethylenediaminetetraacetic acid (EDTA),also known as edetic acid, and diethylenetriaminepentaacetic acid(DTPA), also known as pentetic acid, are shown for comparison since thetwo are considered the ‘gold standard’ metal chelators. The ability tochelate metal cations of acetylcysteine is greatly subpar in comparisonto EDTA and DTPA.

TABLE 2 Complex formation constant (log K) Metal cation EDTA DTPAAcetylcysteine Experimental conditions for Acetylcysteine Fe³⁺ 25.1028.60 10.58 25° C. and 0.12 mol · dm⁻³ ionic strength (NaClO₄) Cu²⁺18.80 21.53 9.02 25° C. and 0.1 mol · dm⁻³ ionic strength (KNO₃) 6.6437° C. and 0.15 mol · dm⁻³ ionic strength (NaCl) Ni²⁺ 18.62 20.32 5.0225° C. and 0.12 mol · dm⁻³ ionic strength (NaClO₄) 4.86 37° C. and 0.15mol · dm⁻³ ionic strength (NaCl) Zn²⁺ 16.50 18.75 7.04 25° C. and 0.1mol · dm⁻³ ionic strength (KNO₃) 6.23 37° C. and 0.15 mol · dm⁻³ ionicstrength (NaCl) Co²⁺ 16.31 18.40 4.18 25° C. and 0.12 mol · dm⁻³ ionicstrength (NaClO₄) 4.28 37° C. and 0.15 mol · dm⁻³ ionic strength (NaCl)Cr³⁺ 23.40 — 8.26 25° C. and 0.12 mol · dm⁻³ ionic strength (NaClO₄)Cd²⁺ 16.46 19.31 7.25 25° C. and 0.1 mol · dm⁻³ ionic strength (KNO₃)Hg²⁺ 21.80 27.00 11.23 25° C. and 0.1 mol · dm⁻³ ionic strength (KNO₃)Mn²⁺ 14.04 15.60 3.64 37° C. and 0.15 mol · dm⁻³ ionic strength (NaCl)Metal complexation in the presence of molecular oxygen promotesoxidation of acetylcysteine in aqueous solutions. As already notedabove, the inventors therefore contemplate that it might be beneficialto include excipients with stronger chelation potency thanacetylcysteine, such as EDTA and DTPA, in the formulation tocompetitively sequester metal cations. Both EDTA and DTPA are expectedto have stronger metal complex equilibrium (would likely retain themetal bounded) to mask the metal from reacting with molecular oxygen andother radical promoting species.

Based on the above and further considerations, the inventors thereforecontemplate that aqueous formulations with ready-to-administeracetylcysteine concentrations will generally fall within a relativelynarrow range of pH and will further avoid a variety of componentsotherwise commonly used in pharmaceutical formulations. Moreover,contemplated formulations will preferably contain one or more chelatingagents at relatively low concentrations, which will also not interferewith chemical stability of acetylcysteine.

For example, an exemplary formulation according to the inventive subjectmatter will include acetylcysteine at a concentration of 25 mg/mL or 50mg/mL and will further include EDTA as a chelator at a concentration ofbetween 10 and 60 mcg/mL. Most typically, such formulations are preparedusing water for injection and the pH will be adjusted with NaOH to pH7.0. Advantageously, such formulations will not require any antioxidantand buffer.

With respect to contemplated acetylcysteine concentrations it istypically preferred that the acetylcysteine is present in theformulation at a final concentration of at least 1 mg/mL, or at least 5mg/mL, or at least 10 mg/mL, or at least 25 mg/mL, or at least 50 mg/mL,or at least 75 mg/mL, or at least 100 mg/mL, or even higher. Therefore,suitable concentration ranges for acetylcysteine in the finalformulation will be between 1 and 10 mg/mL, or between 10 and 40 mg/mL,or between 25 and 75 mg/mL, or between 50 and 100 mg/mL, or even higher.Viewed from a different perspective, suitable acetylcysteineconcentrations will be 10 mg/mL, or 25 mg/mL, or 50 mg/mL, or 75 mg/mL,or 100 mg/mL, and even higher. Thus, it should be recognized that theacetylcysteine in the final formulation will have a concentration thatis suitable for injection without further need for dilution. It shouldbe noted that these and all other concentrations mentioned herein mayalso be approximate concentrations that have a variability of ±5%, or±10%, or ±15%, or ±20% of the given value.

As already noted above, the acetylcysteine concentrations contemplatedherein will generally have an acidic pH which will generally be equal orless than 7.5, or equal or less than 7.0, or equal or less than 6.5, orequal or less than 6.0, or equal or less than 5.5, and in some caseseven less. Thus, contemplated pH ranges will typically be between 5.5and 6.5, or between 6.0 and 7.0, or between 6.5 and 7.5. For example,contemplated formulations may have a pH of 5.5±0.2, or may have a pH of5.8±0.2, or may have a pH of 6.0±0.2, or may have a pH of 6.3±0.2, ormay have a pH of 6.5±0.2, or may have a pH of 6.8±0.2, or may have a pHof 7.0±0.2, or may have a pH of 7.2±0.2.

While it is generally preferred that contemplated formulations will beadjusted to a desirable pH without use of a buffer, various buffers andbuffer systems including organic, inorganic, and amphoteric buffers arenevertheless deemed suitable for use herein. Therefore, especiallycontemplated buffer systems include phosphate buffers MOPS, HEPES, MES,Bis-TRIS, etc. Most typically, and while not limiting to the inventivesubject matter, the inventors contemplate that the buffer strengthrequired for stabilization of acetylcysteine will be relatively low, forexample, equal or less than 100 mM, and more typically equal or lessthan 50 mM, and most typically between 5 mM and 20 mM (e.g., 10 mM).therefore, contemplated buffers will generally have a concentration ofbetween about 0.1-1 mM, or between about 1-5 mM, or between about 5-10mM, or between about 10-20 mM, or between about 2-50 mM, and in somecases higher.

In still further contemplated aspects, the formulations presented hereinwill also include one or more chelating agents, and particularly metalion chelators. For example, suitable chelators include variousbicarboxylic acids, tricarboxylic acids, and aminopolycarboxylic acidssuch as ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), andpenta(carboxymethyl)diethylenetriamine (DTPA), and salts and hydratesthereof. While not limiting to the inventive subject matter, it iscontemplated that the metal ion chelators will slow down both thebaseline and metal ion-stimulated degradation of acetylcysteine.Remarkably, the inventors unexpectedly observed that the desirableeffect of the chelators was observable at relatively low concentrationsof the chelators. For example, reduction of the baseline and metalion-stimulated degradation of acetylcysteine was observed at chelatorconcentrations of between 1 mcg/ml and 10 mcg/ml, between 10 mcg/ml and30 mcg/ml, between 30 mcg/ml and 50 mcg/ml, between 50 mcg/ml and 70mcg/ml, or between 70 mcg/ml and 100 mcg/ml. Interestingly, thechelators, and especially the aminopolycarboxylic acids retainedstabilizing effect despite the relatively low pH favoring protonatedforms of the chelators. In further embodiments, the chelator may also beomitted, especially where compounding will avoid use of metalcontainers.

In yet further preferred aspects, it should be appreciated thatcontemplated formulations will not require an antioxidant. However, itshould be noted that one or more antioxidants could be included, andparticularly preferred antioxidants include cysteine hydrochloride,methionine, ascorbic acid, tocopherol, and sodium metabisulfite. Mostantioxidants are deemed to have stabilizing effect even when used atrelatively low concentrations, and preferred concentrations includethose between 0.1-5 mcg/mL, or between 5-10 mcg/mL, or between 10-30mcg/mL, or between 30-50 mcg/mL, or between 50-100 mcg/mL. Thus,contemplated antioxidant concentrations may be 1 mcg/mL, or 5 mcg/mL, or10 mcg/mL, or 25 mcg/mL, or 50 mcg/mL, or 100 mcg/mL.

With respect tonicity agents, it is generally contemplated that allknown tonicity agents can be used in conjunction with the teachingspresented herein and suitable tonicity agents include variouscarbohydrates (e.g., dextrose, mannitol, etc.), glycols (e.g.,glycerol), and pharmaceutically acceptable salts (e.g., NaCl). Theamount of tonicity adjusting agent used can be adjusted to obtain anosmolality of the formulations in the range of 260 to 340 mOsm/kg. Anosmometer can be used to check and determine or adjust the amount oftonicity adjusting agent to be added to obtain the desired osmolality.

Notably, contemplated acetylcysteine formulations were stable asdescribed in more detail below, especially where deoxygenated solvents(e.g., typically water and/or buffer) were employed. Deoxygenation(i.e., reduction of molecular dissolved oxygen) can be achieved innumerous manners, including sparging with inert gases (e.g., helium,various freons, argon, xenon), agitation under vacuum, and/or usingenzymatic systems that deplete a solution of dissolved oxygen (see e.g.,U.S. Pat. No. 9,187,779). Moreover, it is generally preferred that anyheadspace in the container will be maintained under an oxygen-reduced ordepleted state. Additionally, or alternatively, ingress of molecularoxygen into the formulation can also be reduced by co-packaging acontainer with the formulation in a secondary container that includes anoxygen scavenger, and especially a metal-free oxygen scavenger (e.g.,GLS100, Ageless®, Pharmakeep®, all commercially available fromMitsubishi Gas Chemical America). Therefore, it should be noted thatnon-contact oxygen scavengers are especially preferred.

With respect to the sterilization of contemplated formulations it shouldbe appreciated that contemplated formulations may be sterilized usingall known manners of sterilization, including filtration through 0.22micron filters, heat sterilization, autoclaving, radiation (e.g., gamma,electron beam, microwave), and gas sterilization (e.g., using ethyleneoxide gas). Based on the anticipated moderate stability, theformulations contemplated herein are filtered through a 0.22-micron (orother suitably sized) filter, and then filled in to a container such asa polyethylene, polypropylene, or low-density polyethylene container.Where desired, the filling may include a blow-fill-seal (BFS) process.

BFS is a form of advanced aseptic manufacturing wherein the container isformed, filled, and sealed in one continuous, automated system notrequiring human intervention. The process begins with the extrusion ofplastic granules in the form of a hot hollow pipe of molten plasticcalled a parison. The next step is the blow molding of the containerwith an open top through which the container is filled, all while theplastic remains hot and in a molten state. Once filled, the container ishermetically sealed and cooled. The blow-fill seal process can takeseveral seconds, and contemplated ready-to-inject compositionsadvantageously are formulated to withstand the temperature and pressurerequirements without substantial degradation of acetylcysteine (e.g.,less than 5 wt %, less than 3 wt %, less than 2 wt %, less than 1 wt %degradation).

Once the acetylcysteine formulations are filled in large volumepolymeric, semi-permeable infusion containers (e.g., BFS container orflexible IV bags), the containers can optionally be layered or coveredwith a secondary packaging system optionally including an oxygenscavenger. For example, the BFS containers can further be sealed in anoxygen and moisture barrier blister packaging. The blister packaging cancomprise one or more layers, and the one or more layers can includealuminum foil or other oxygen absorber having an Oxygen TransmissionRate (OTR) between 0.0005 to 5.00 cc/100 in²/24 hrs. Additionally oralternatively, one or more oxygen absorbers (metal or metal free,organic material) can be incorporated into any portion of the BFScontainer, the secondary packaging system, or between the two (e.g.,between the BFS container and the multi-layer packaging) such that theoxygen absorber removes at least a portion of oxygen from the airsurrounding said oxygen-sensitive drug. A beneficial feature of theoxygen absorber is the absorbance and removal of oxygen present in theprimary packaging and in the liquid drug itself. Notably, it was foundthat the oxygen absorber also removed residual headspace oxygen in theprimary packaging and also dissolved oxygen in the liquid over time,thereby further improving stability of acetylcysteine. In still furthercontemplated but less preferred aspects, the container may also be aglass container.

As is described in more detail below, contemplated compositions exhibitsignificant storage stability over extended periods of time, even wherethe storage is at ambient (25° C.) or significantly higher temperaturessuch as 40° C., or 60° C., or even 80° C. In most cases, stability canbe expressed as the presence of total impurities where the impuritiesare generated from the degradation of the acetylcysteine via one or moredegradation pathways. For example, contemplated composition willtypically have a storage stability at ambient temperature (25° C.) of atleast 6 months, or at least 12 months, or at least 18 months, or atleast 24 months, after which equal or less than 10%, or equal or lessthan 8%, or equal or less than 7%, or equal or less than 6%, or equal orless than 5%, or equal or less than 4%, or equal or less than 3% oftotal impurities are present are generated from the degradation of theacetylcysteine. Similarly, it is contemplated that contemplatedcomposition will typically have a storage stability at elevated storagetemperature (40 or 60° C.) of at least 3 months, or at least 6 months,or at least 12 months, or at least 18 months, after which equal or lessthan 10%, or equal or less than 8%, or equal or less than 7%, or equalor less than 6%, or equal or less than 5%, or equal or less than 4%, orequal or less than 3% of total impurities are present are generated fromthe degradation of the acetylcysteine. Likewise, it is contemplated thatcontemplated composition will typically have a storage stability at highultra-high storage temperature (80° C.) of at least 2 weeks, or at least3 weeks, or at least 4 weeks, or at least 6 weeks, or at least 12 weeks,after which equal or less than 10%, or equal or less than 8%, or equalor less than 7%, or equal or less than 6%, or equal or less than 5%, orequal or less than 4%, or equal or less than 3% of total impurities arepresent are generated from the degradation of the acetylcysteine. Ofcourse, it should be appreciated that the compositions presented hereinwill exceed under refrigeration (4-8° C.) the stability otherwiseobserved for storage 12 months, or at least 18 months, or at least 24months at ambient temperature.

In yet further contemplated aspects, the acetylcysteine compositionspresented herein are preferably packaged in a container in an amountthat provides a quantity sufficient for administration of at least onedose of a multi-dose regimen. Therefore, and viewed from a differentperspective, the volume of ready-to-administer acetylcysteine will besufficient to allow for administration of more than a single dose of amulti-dose regimen. Therefore, suitable quantities in a single containerwill be between 100 and 200 mL, or between 150 and 250 mL, or between200 and 300 mL, or between 250 and 500 mL, or between 300 and 600 mL, oreven higher. For example, the volume of ready-to-administeracetylcysteine in a single container may be 50 mL, 100 mL, 150 mL, 200mL, 250 mL, 300 mL, or 500 mL, or even higher. The acetylcysteineconcentration in such containers will preferably be 25 mg/mL or 50 mg/mL(however, other concentrations as discussed above are also deemedappropriate).

In still further contemplated embodiments, acetylcysteine will beadministered in a multi-dose regimen comprising a first (loading) dose,a second (maintenance) dose, and a third (maintenance dose), with thesecond and third doses typically delivering distinct quantities overdistinct periods of administration. Exemplary doses and schedules ofadministration are depicted in Table 3 (pediatric use) and Table 4(non-pediatric use). The values given in the different doses indicatethe amount of acetylcysteine administered with the corresponding volumelisted in parentheses. As can be readily seen form the Tables, theconcentration of acetylcysteine is constant over all three doses, andthe quantity of acetylcysteine over a given time can be readily using apredetermined rate of administration (which will typically be performedvia a rate-controlled infusion pump). Moreover, and especially forsubjects with normal o lower body weight, it should be recognized thatthe overall fluid load is significantly lower as compared toadministration of the same quantity of acetylcysteine when diluted froma concentrate.

TABLE 3 Body Bag 1 (Loading Dose) Bag 2 (Second Dose) Bag 3 (Third Dose)Weight Concentration infused over 1 hour infused over 4 hours infusedover 16 hours  5 kg 50 mg/mL 750 mg (15 mL) 250 mg (5 mL) 500 mg (10 mL)10 kg 50 mg/mL 1,500 mg (30 mL) 500 mg (10 mL) 1,000 mg (20 mL) 15 kg 50mg/mL 2,250 mg (45 mL) 750 mg (15 mL) 1,500 mg (30 mL) 20 kg 50 mg/mL3,000 mg (60 mL) 1,000 mg (20 mL) 2,000 mg (40 mL)

TABLE 4 Body Bag 1 (Loading Dose) Bag 2 (Second Dose) Bag 3 (Third Dose)Weight Concentration infused over 1 hour infused over 4 hours infusedover 16 hours   21 kg 25 mg/mL 3,150 mg (126 mL) 1,050 mg (42 mL) 2,100mg (84 mL)   30 kg 25 mg/mL 4,500 mg (180 mL) 1,500 mg (60 mL) 3,000 mg(120 mL)   40 kg 25 mg/mL 6,000 mg (240 mL) 2,000 mg (80 mL) 4,000 mg(160 mL)   41 kg 50 mg/mL 6,150 mg (123 mL) 2,050 mg (41 mL) 4,100 mg(82 mL)   50 kg 50 mg/mL 7,500 mg (150 mL) 2,500 mg (50 mL) 5,000 mg(100 mL)   60 kg 50 mg/mL 9,000 mg (180 mL) 3,000 mg (60 mL) 6,000 mg(120 mL)   70 kg 50 mg/mL 10,500 mg (210 mL) 3,500 mg (70 mL) 7,000 mg(140 mL)   80 kg 50 mg/mL 12,000 mg (240 mL) 4,000 mg (80 mL) 8,000 mg(160 mL)   90 kg 50 mg/mL 13,500 mg (270 mL) 4,500 mg (90 mL) 9,000 mg(180 mL) ≥100 kg 50 mg/mL 15,000 mg (300 mL) 5,000 mg (100 mL) 10,000 mg(200 mL)

EXAMPLES

The following examples are intended as representative embodiments of theinventive subject matter and should not be construed as limiting thescope of the invention.

Storage stability of acetylcysteine was evaluated at pH 6.0, 6.5, 7.0,7.5 and 8.0. To that end, batches were manufactured in glass containers,and nitrogen sparging was performed on every step to ensure low levelsof oxygen. All solutions were dispensed in polypropylene bags and werepackaged (vacuum-sealed) in a secondary aluminum overwrap containing anon-contact oxygen scavenger. Samples were stored at 80° C. for up to 3weeks. Tables 5-9 show exemplary results. As can be readily seen formthe data, acetylcysteine in the tested formulations had desirablestability over a pH range of 6.0 to 7.5. Such observation was unexpectedat the very low tested concentrations for acetylcysteine, particularlyin the absence of buffer, other stabilizing agents, and antioxidants.However, acetylcysteine degradation was relatively higher at pH 8.0.Therefore, further evaluations were performed at pH 7.0.

TABLE 5 Lot # 14003-1 Stability data for 25 mg/mL Acetylcysteine at pH6.0 (80° C.) Time point Initial 1 Day 4 Day 1 Week 2 Week 3 WeekAppearance CCS CCS CCS CCS CCS CCS pH 6.81 6.56 6.40 6.52 6.29 6.23Dissolved Oxygen 1.34 1.03 0.96 0.97 0.97 0.98 (ppm) Osmolality 295 298302 300 300 300 (mOsm/kg) Assay (%) 98.6 100.5 94.4 96.0 95.4 95.5Related RC-A NR NR NR NR NR NR substance RC-B 0.24 0.56 0.90 1.21 1.752.40 (%) RC-C 0.45 0.59 1.48 0.81 0.86 1.47 RC-D 0.27 0.22 0.15 0.130.10 0.09 Unknown NR 0.06 (0.94) NR (0.30) NR (0.30) 0.14 (0.30) 0.31(0.30) (RRT) NR (1.06) 0.07 (0.41) NR (0.50) 0.10 (0.50) NR (0.46) NR(0.50) 0.31 (0.94) 0.61 (0.94) 0.16 (0.50) 0.19 (0.94) NR (1.06) NR(1.06) 0.99 (0.94) NR (1.06) 0.06 (1.21) 0.16 (1.21) NR (1.06) NR (1.21)NR (1.38) 0.05 (1.38) 0.33 (1.21) NR (1.38) NR (1.54) 0.05 (1.54) NR(1.35) NR (1.54) NR (1.60) NR (1.60) 0.07 (1.38) NR (1.60) NR (1.44)0.09 (1.54) 0.08 (1.60) Total Impurity (%) 0.95 1.43 2.78 2.53 3.82 6.02

TABLE 6 Lot # 14003-2 Stability data for 25 mg/mL Acetylcysteine at pH6.5 (80° C.) Time point Initial 1 Day 4 Day 1 Week 2 Week 3 WeekAppearance CCS CCS CCS CCS CCS CCS pH 6.94 6.75 6.60 6.52 6.46 6.39Dissolved Oxygen 1.09 0.90 1.01 0.86 0.94 0.90 (ppm) Osmolality 294 295300 299 298 300 (mOsm/kg) Assay (%) 99.3 101.1 96.1 97.1 95.6 94.9Related RC-A NR NR NR NR NR NR substance RC-B 0.22 0.44 0.89 1.16 1.732.28 (%) RC-C 0.34 0.43 0.52 0.47 0.78 1.09 RC-D 0.22 0.18 0.10 0.090.07 0.07 Unknown NR NR (0.94) NR (0.30) 0.05 (0.30) 0.14 (0.30) 0.30(0.30) (RRT) NR (1.06) NR (0.50) NR (0.50) 0.09 (0.50) 0.15 (0.50) 0.20(0.94) 0.34 (0.94) 0.65 (0.94) NR (0.80) NR (1.06) NR (1.06) NR (1.06)1.00 (0.94) NR (1.21) 0.07 (1.21) 0.20 (1.21) NR (1.06) NR (1.54) NR(1.38) NR (1.38) 0.37 (1.21) NR (1.60) NR (1.54) 0.06 (1.54) NR (1.35)NR (1.60) 0.06 (1.60) 0.07 (1.38) NR (1.44) 0.13 (1.54) NR (1.57) 0.09(1.60) Total Impurity (%) 0.77 1.04 1.71 2.19 3.79 5.57

TABLE 7 Lot # 14003-3 Stability data for 25 mg/mL Acetylcysteine atpH7.0 (80° C.) Time point Initial 1 Day 4 Day 1 Week 2 Week 3 WeekAppearance CCS CCS CCS CCS CCS CCS pH 7.16 7.03 7.01 6.87 6.80 6.81Dissolved Oxygen 0.95 0.94 1.00 0.99 0.86 0.99 (ppm) Osmolality 297 297300 299 301 298 (mOsm/kg) Assay (%) 99.5 101.1 95.3 97.3 95.8 96.5Related RC-A NR NR NR NR NR NR substance RC-B 0.19 0.42 0.69 1.01 1.451.91 (%) RC-C 0.41 0.42 0.52 0.56 1.04 1.18 RC-D 0.16 0.10 NR NR NR NRUnknown NR NR (0.94) NR (0.30) NR (0.30) 0.12 (0.30) 0.27 (0.30) (RRT)NR (1.06) NR (0.50) NR (0.50) 0.08 (0.50) 0.15 (0.50) 0.21 (0.94) 0.33(0.94) NR (0.80) NR (0.71) NR (1.06) NR (1.06) 0.66 (0.94) NR (0.80) NR(1.21) 0.07 (1.21) NR (1.06) 1.07 (0.94) NR (1.38) NR (1.38) 0.21 (1.21)NR (1.06) NR (1.54) NR (1.54) NR (1.38) 0.43 (1.21) NR (1.60) NR (1.60)0.10 (1.54) 0.06 (1.35) 0.10 (1.60) 0.06 (1.38) NR (1.44) 0.19 (1.54)0.16 (1.60) Total Impurity (%) 0.77 0.94 1.42 1.98 3.76 5.47

TABLE 8 Lot # 14003-4 Stability data for 25 mg/mL Acetylcysteine at pH7.5 (80° C.) Time point Initial 1 Day 4 Day 1 Week 2 Week 3 WeekAppearance CCS CCS CCS CCS CCS CCS pH 7.65 7.49 7.49 7.37 7.32 7.30Dissolved Oxygen 1.05 0.85 1.00 0.92 0.82 1.00 (ppm) Osmolality 296 296300 300 303 296 (mOsm/kg) Assay (%) 99.7 100.9 96.3 97.4 95.4 94.3Related RC-A NR NR NR NR NR NR substance RC-B 0.11 0.39 0.65 0.92 1.211.46 (%) RC-C 0.64 0.70 0.92 0.54 1.15 1.61 RC-D 0.11 NR NR NR NR NRUnknown NR NR (0.94) NR (0.30) NR (0.30) 0.11 (0.30) 0.24 (0.30) (RRT)NR (1.06) NR (0.50) NR (0.50) 0.09 (0.50) 0.15 (0.50) 0.22 (0.94) 0.38(0.94) NR (0.80) NR (0.71) NR (1.06) NR (1.06) 0.76 (0.94) 0.05 (0.80)NR (1.21) 0.10 (1.21) NR (1.06) 1.21 (0.94) NR (1.38) NR (1.38) 0.29(1.21) NR (1.06) 0.05 (1.54) 0.11 (1.54) NR (1.34) 0.55 (1.21) 0.05(1.60) 0.10 (1.60) 0.27 (1.54) 0.12 (1.35) 0.24 (1.60) NR (1.38) 0.42(1.54) 0.37 (1.60) Total Impurity (%) 0.86 1.09 1.89 2.15 4.13 6.18

TABLE 9 Lot # 14003-5 Stability data for 25 mg/mL Acetylcysteine at pH8.0 (80° C.) Time point Initial 1 Day 4 Day 1 Week 2 Week 3 WeekAppearance CCS CCS CCS CCS CCS CCS pH 8.05 7.95 7.92 7.89 7.83 7.84Dissolved Oxygen 1.02 0.90 0.93 0.88 0.93 0.88 (ppm) Osmolality 298 296297 300 301 293 (mOsm/kg) Assay (%) 99.9 100.5 91.4 96.4 92.4 94.1Related RC-A NR NR NR NR NR NR substance RC-B 0.10 0.49 0.56 0.87 0.971.09 (%) RC-C 0.52 0.69 4.81 0.79 2.72 1.55 RC-D 0.06 NR NR NR NR NRUnknown NR 0.08 (0.94) NR (0.30) NR (0.30) 0.09 (0.30) 0.18 (0.30) (RRT)NR (1.06) 0.26 (0.41) 0.05 (0.50) 0.11 (0.41) NR (0.33) NR (0.50) 0.42(0.94) 0.12 (0.50) NR (0.46) 0.25 (0.94) NR (1.06) NR (0.71) 0.19 (0.50)NR (1.06) 0.12 (1.21) NR (0.80) 0.06 (0.71) 0.05 (1.21) 0.24 (1.54) 0.92(0.94) 0.07 (0.80) 0.11 (1.54) 0.24 (1.60) NR (1.06) 1.51 (0.94) 0.11(1.60) 0.36 (1.21) NR (1.06) 0.10 (1.34) 0.69 (1.21) 0.63 (1.54) NR(1.47) 0.59 (1.60) 1.09 (1.54) 0.92 (1.60) NR (1.67) Total Impurity (%)0.68 1.27 6.15 2.73 6.6 7.35

The inventors then investigated the effect of a metal chelator inacetylcysteine formulations. To that end, samples were prepared at pH7.0 that included edetate disodium dihydrate (EDTA) at concentrations ofeither 0 mcg/mL (control), 10 mcg/mL, 20 mcg/mL, 30 mcg/mL, 40 mcg/mL,and 60 mcg/mL. Once again, the samples were manufactured in glasscontainers, and nitrogen sparging was performed on every step to ensurelow levels of oxygen. Solutions were dispensed in polypropylene bags andwere packaged (vacuum-sealed) in a secondary aluminum overwrapcontaining a non-contact oxygen scavenger. The samples were stored ateither 60° C. for up to 2 weeks or at 80° C. for up to 1 week asindicated, and exemplary results are shown in Tables 10-15. While theeffects did not reveal a trend or change in stability, it should benoted that the chelator also did not adversely affect stability at thetested low concentrations. In that regard, it is contemplated that wheremanufacture is modified to include steel containers, metal leakage andassociated metal-catalyzed degradation may occur unless a chelator isemployed.

TABLE 10 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing0 mcg/mL EDTA (control) Lot #13991-1 60° C. 80° C. Time point Initial 4Day 1 Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCSCCS pH 7.18 7.01 7.04 6.96 7.01 6.87 6.85 Dissolved Oxygen 1.01 1.050.96 1.07 0.96 0.96 0.90 (ppm) Osmolality 298 296 296 296 295 300 297(mOsm/kg) Assay (%) 98.5 97.9 99.1 100.4 100.2 95.7 98.2 Related RC-A NRNR NR NR NR NR NR substance RC-B 0.18 0.23 0.37 0.56 0.42 0.72 1.15 (%)RC-C 0.32 0.33 0.31 0.34 0.56 0.53 0.48 RC-D 0.17 0.12 0.10 0.07 0.110.05 NR Unknown NR NR (0.94) NR (0.94) NR (0.50) NR (0.94) NR (0.30)0.05 (0.30) (RRT) 0.07 (0.94) NR (1.06) NR (0.50) NR (0.50) NR (1.06)0.21 (0.94) 0.38 (0.94) NR (1.06) NR (1.06) NR (1.21) 0.10 (1.21) NR(1.60) NR (1.38) 0.05 (1.54) 0.06 (1.60) Total Impurity (%) 0.67 0.670.78 1.04 1.09 1.52 2.27

TABLE 11 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing10 mcg/mL EDTA Lot #13991-2 60° C. 80° C. Time point Initial 4 Day 1Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCS CCS pH7.28 7.21 7.25 7.22 7.22 7.08 7.23 Dissolved Oxygen 0.94 0.95 0.91 1.040.92 0.97 0.96 (ppm) Osmolality 293 294 295 293 293 298 294 (mOsm/kg)Assay (%) 98.2 96.8 98.7 100.3 99.1 94.6 97.2 Related RC-A NR NR NR NRNR NR NR substance RC-B 0.18 0.26 0.36 0.48 0.40 0.80 1.03 (%) RC-C 0.200.39 0.32 0.29 0.39 0.43 0.44 RC-D 0.14 0.08 0.06 0.07 0.07 NR NRUnknown NR NR (0.94) NR (0.94) NR (0.50) NR (0.94) NR (0.30) 0.05 (0.30)(RRT) NR (1.06) 0.08 (0.94) NR (1.06) NR (0.50) NR (0.50) NR (1.06) 0.22(0.94) 0.40 (0.94) NR (1.21) NR (1.06) NR (1.06) NR (1.21) 0.11 (1.21)NR (1.38) NR (1.38) NR (1.44) 0.07 (1.54) NR (1.54) 0.07 (1.60) NR(1.60) Total Impurity (%) 0.53 0.74 0.74 0.86 1.09 1.52 2.27

TABLE 12 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing20 mcg/mL EDTA Lot #13991-2 60° C. 80° C. Time point Initial 4 Day 1Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCS CCS pH7.24 7.10 7.20 7.08 7.17 6.96 7.01 Dissolved Oxygen 0.90 1.02 0.98 1.150.87 0.88 0.88 (ppm) Osmolality 298 301 297 301 299 297 300 (mOsm/kg)Assay (%) 99.4 98.2 100.2 101.6 100.8 97.2 97.8 Related RC-A NR NR NR NRNR NR NR substance RC-B 0.18 0.27 0.38 0.54 0.44 0.86 1.07 (%) RC-C 0.400.40 0.38 0.38 0.50 0.55 0.52 RC-D 0.16 0.10 0.08 0.06 0.09 NR NRUnknown NR NR (0.94) NR (0.94) NR (0.50) NR (0.94) NR (0.30) 0.06 (0.30)(RRT) 0.08 (0.94) NR (1.06) NR (0.50) 0.05 (0.50) NR (1.06) 0.23 (0.94)0.42 (0.94) NR (1.21) NR (1.06) NR (1.06) 0.05 (1.21) 0.11 (1.21) NR(1.38) NR (1.38) NR (1.44) 0.06 (1.54) NR (1.54) 0.06 (1.60) NR (1.60)Total Impurity (%) 0.74 0.76 0.84 1.06 1.04 1.69 2.36

TABLE 13 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing30 mcg/mL EDTA Lot# 13991-4 60° C. 80° C. Time point Initial 4 Day 1Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCS CCS pH7.20 7.17 7.22 7.09 7.12 6.98 7.03 Dissolved Oxygen 1.08 0.99 0.85 1.170.87 0.93 0.96 (ppm) Osmolality 298 299 301 290 296 296 296 (mOsm/kg)Assay (%) 99.3 96.7 100.4 101.9 101.2 97.4 98.4 Related RC-A NR NR NR NRNR NR NR substance RC-B 0.17 0.27 0.37 0.56 0.40 0.86 1.11 (%) RC-C 0.200.14 0.23 0.19 0.25 0.26 0.32 RC-D 0.16 0.10 0.08 0.06 0.09 NR NRUnknown NR NR (0.94) NR (0.94) NR (0.50) NR (0.94) NR (0.30) 0.06 (0.30)(RRT) 0.07 (0.94) NR (1.06) NR (0.50) NR (0.50) NR (1.06) 0.23 (0.94)0.41 (0.94) NR (1.06) NR (1.06) NR (1.21) 0.11 (1.21) NR (1.38) NR(1.38) NR (1.44) 0.06 (1.54) NR (1.54) 0.06 (1.60) NR (1.60) TotalImpurity (%) 0.53 0.51 0.69 0.88 0.74 1.35 2.12

TABLE 14 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing40 mcg/mL EDTA Lot #13991-5 60° C. 80° C. Time point Initial 4 Day 1Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCS CCS pH7.15 7.10 7.16 7.00 7.09 6.93 7.01 Dissolved Oxygen 0.98 1.03 0.98 1.001.00 0.85 0.93 (ppm) Osmolality 301 300 296 298 298 300 299 (mOsm/kg)Assay (%) 99.4 98.1 99.8 102.0 100.9 95.7 97.9 Related RC-A NR NR NR NRNR NR NR substance RC-B 0.19 0.29 0.38 0.58 0.42 0.84 1.07 (%) RC-C 0.220.23 0.21 0.18 0.35 0.32 0.38 RC-D 0.16 0.11 0.09 0.07 0.10 NR NRUnknown NR NR (0.94) NR (0.94) NR (0.50) NR (0.94) NR (0.30) 0.06 (0.30)(RRT) 0.07 (0.94) NR (1.06) NR (0.50) NR (0.50) NR (1.06) 0.22 (0.94)0.39 (0.94) NR (1.21) NR (1.06) NR (1.06) NR (1.21) 0.10 (1.21) NR(1.38) NR (1.38) NR (1.54) 0.05 (1.54) NR (1.60) NR (1.60) TotalImpurity (%) 0.57 0.63 0.68 0.90 0.88 1.38 2.05

TABLE 15 Stability data for 25 mg/mL Acetylcysteine at pH 7.0 containing60 mcg/mL EDTA Lot #13991-6 60° C. 80° C. Time point Initial 4 Day 1Week 2 Week 1 Day 4 Day 1 Week Appearance CCS CCS CCS CCS CCS CCS CCS pH7.17 7.10 7.22 7.08 7.09 6.98 7.06 Dissolved Oxygen 1.05 1.07 0.92 0.951.01 0.89 0.91 (ppm) Osmolality 296 302 298 296 297 301 302 (mOsm/kg)Assay (%) 99.4 98.0 99.9 102.2 100.6 95.1 96.5 Related RC-A NR NR NR NRNR NR NR substance RC-B 0.18 0.36 0.37 0.58 0.41 0.85 1.08 (%) RC-C 0.260.29 0.29 0.34 0.44 0.50 0.68 RC-D 0.16 0.10 0.08 0.06 0.10 NR NRUnknown NR NR (0.94) NR (0.94) 0.08 (0.94) NR (0.94) NR (0.30) 0.06(0.30) (RRT) NR (1.06) NR (1.06) NR (0.50) NR (0.50) NR (1.21) 0.24(0.94) 0.42 (0.94) NR (1.06) 0.11 (1.21) NR (1.21) NR (1.38) NR (1.38)0.06 (1.54) NR (1.54) 0.06 (1.60) NR (1.60) Total Impurity (%) 0.60 0.750.74 1.06 0.94 1.60 2.48

In still further experiments, the inventors sought to determine thefeasibility of terminal sterilization (here: autoclaving) via an airoverpressure (AOP) cycle at 121° C. for either 15-mins or 30-minsexposure time (ET). Acetylcysteine solutions were prepared at pH 7.0with or without EDTA (as indicated in above). Samples were manufacturedin glass containers, and nitrogen sparging was performed on every stepto ensure low levels of oxygen. Solutions were dispensed inpolypropylene bags. Accordingly, bags were either autoclaved before(Table 16) or after packaging (Table 17), unless otherwise indicated.Bags handled following the former sterilization process were then cooledafter autoclaving and packaged (vacuum-sealed) in a secondary aluminumoverwrap containing a non-contact oxygen scavenger. Bags following thelater sterilization process were packaged (vacuum-sealed) initially insecondary aluminum overwrap containing an autoclavable non-contactoxygen scavenger and held at room temperature for 2 days prior toautoclaving. No further packaging steps were performed afterautoclaving. In this context, it should be noted that the overwrap neednot necessarily be metallized or otherwise light impermeable.

Bags that were not autoclaved were packaged similarly to previousstudies, where bags were encased (vacuum-sealed) in a secondary aluminumoverwrap containing a non-contact oxygen scavenger. As can be seen formthe results in Tables 16 and 17, the impurity levels in autoclavedacetylcysteine solutions were higher as compared to not-autoclaved(aseptically filled) batches (Table 16). Notably, it appeared that thesterilization process in which the bags were packaged prior toautoclaving (Table 17) resulted in lower impurity levels as compared tothe sterilization process wherein bare bags were autoclaved (Table 16).However, both sterilization techniques yielded higher impurity levels ascompared to aseptic filling (preferably performed using sterilefiltration).

TABLE 16 25 mg/mL Acetylcysteine at pH 7.0 containing either 20 mcg/mLor 60 mcg/mL EDTA and either autoclaved (before packaging) or notautoclaved Amount of EDTA in formulation 20 mcg/mL EDTA (Lot # 13992-1)60 mcg/mL EDTA (Lot # 13992-4) Not Not Condition autoclaved 15 mins ET30 mins ET autoclaved 15 mins ET 30 mins ET Appearance CCS CCS CCS CCSCCS CCS pH 7.22 6.98 7.06 7.25 7.06 7.06 Dissolved Oxygen 0.92 1.18 1.090.95 1.03 1.04 (ppm) Osmolality 296 296 296 349*    347*    347*   (mOsm/kg) Assay (%) 102.4 101.9 99.4 103.0   99.0  99.1  Related RC-A NRNR NR NR NR NR substance RC-B 0.18 0.35 0.37 0.20 0.33 0.40 (%) RC-C0.80 1.95 2.00 0.34 1.36 1.46 RC-D 0.15 0.12 0.13 0.16 0.12 0.12 UnknownNR 0.05 (0.94) 0.06 (0.94) NR (0.33) NR (0.94) 0.08 (0.94) (RRT) 0.06(1.06) 0.08 (1.06) 0.13 (1.12) NR (1.06) 0.15 (1.12) 0.15 (1.12) NR(1.21) 0.23 (1.12) 0.05 (1.21) 0.06 (1.21) NR (1.54) 0.08 (1.21) NR(1.54) NR (1.54) 0.05 (1.54) NR (1.60) NR (1.60) Total Impurity (%) 1.132.73 2.86 0.70 1.95 2.42

TABLE 17 25 mg/mL Acetylcysteine at pH 7.0 containing varying levels ofEDTA and autoclaved after packaging with ATCO HV300M oxygen scavengerLot# 14172 0 mcg/ 10 mcg/ 20 mcg/ 30 mcg/ 40 mcg/ 60 mcg/ mL EDTA mLEDTA mL EDTA mL EDTA mL EDTA mL EDTA Appearance CCS CCS CCS CCS CCS CCSpH 6.94 7.06 7.01 7.03 7.01 7.03 Dissolved Oxygen 1.14 1.28 1.21 1.191.08 1.19 (ppm) Osmolality 295 288 304 298 295 302 (mOsm/kg) Assay (%)100.4 99.2 97.7 99.1 99.9 100.1 Related RC-A NR NR NR NR NR NR substanceRC-B 0.52 0.44 0.47 0.46 0.50 0.43 (%) RC-C 0.53 0.42 0.47 0.30 0.470.50 RC-D 0.14 0.09 0.11 0.11 0.12 0.12 Unknown 0.08 (0.94) 0.09 (0.94)0.07 (0.94) 0.07 (0.94) 0.08 (0.94) 0.09 (0.94) (RRT) 0.23 (1.06) 0.24(1.06) 0.21 (1.06) 0.21 (1.06) 0.22 (1.06) 0.24 (1.06) 0.06 (1.21) 0.06(1.21) 0.06 (1.21) 0.05 (1.21) 0.06 (1.21) 0.07 (1.21) NR (1.54) NR(1.38) NR (1.38) NR (1.38) NR (1.38) NR (1.38) NR (1.60) NR (1.54) NR(1.54) NR (1.54) NR (1.54) NR (1.54) NR (1.60) NR (1.60) NR (1.60) NR(1.60) NR (1.60) NR (1.64) Total Impurity (%) 1.55 1.33 1.40 1.21 1.461.44

Acetylcysteine and its impurities were determined by a reverse phaseHPLC method. The method was modified version of an EP method forAcetylcysteine. The column used was Phenomenex Luna® 5 μm C18(2) 100 Å(column dimension 250×4.6 mm) and a gradient of 0.1% phosphoric acid inwater and acetonitrile was used as a mobile phase for analysis.Quantitation of Acetylcysteine is accomplished by comparingcorresponding peak areas from sample solution chromatogram to that froma Reference Standard (RS) solution of a known concentration. The diluentsolution used comprise of EDTA dissolved in water (0.06 mg/mL) and pHadjusted to 7.0±0.1 with 0.1N sodium hydroxide. Water used to preparethe diluent was degassed to achieve a dissolved oxygen level below 1 ppmand the sample temperature was maintained at 5° C. to prevent theoxidation of Acetylcysteine during analysis.

As used herein, the term “administering” a pharmaceutical composition ordrug refers to both direct and indirect administration of thepharmaceutical composition or drug, wherein direct administration of thepharmaceutical composition or drug is typically performed by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirectadministration includes a step of providing or making available thepharmaceutical composition or drug to the health care professional fordirect administration (e.g., via injection, infusion, oral delivery,topical delivery, etc.). It should further be noted that the terms“prognosing” or “predicting” a condition, a susceptibility fordevelopment of a disease, or a response to an intended treatment ismeant to cover the act of predicting or the prediction (but nottreatment or diagnosis of) the condition, susceptibility and/orresponse, including the rate of progression, improvement, and/orduration of the condition in a subject.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe full scope of the present disclosure, and does not pose a limitationon the scope of the invention otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the claimed invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the full scope of the concepts disclosed herein. Thedisclosed subject matter, therefore, is not to be restricted except inthe scope of the appended claims. Moreover, in interpreting both thespecification and the claims, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced. Where the specification claims refers to atleast one of something selected from the group consisting of A, B, C . .. and N, the text should be interpreted as requiring only one elementfrom the group, not A plus N, or B plus N, etc.

What is claimed is:
 1. A sterile and ready-to-administer N-acetylcysteine composition, comprising: an aqueous solution containing N-acetylcysteine at a concentration suitable for direct administration via intravenous injection to a patient without dilution prior to administration; wherein the composition has a pH of between 6.0 and 7.5 and comprises a chelating agent at a concentration of between 10 and 60 mcg/mL; and wherein the ready-to-administer composition contains, after storage over at least three weeks at 80° C., equal or less than 7% total impurities generated from degradation of the N-acetylcysteine.
 2. The composition of claim 1 wherein the N-acetylcysteine concentration is 25 mg/mL or 50 mg/mL.
 3. The composition of claim 1 wherein the composition has a pH of 7.0±0.2.
 4. The composition of claim 1 wherein composition contains, after storage over at least three weeks at 80° C., equal or less than 6% total impurities generated from degradation of the N-acetylcysteine.
 5. A kit comprising: a primary container that contains a sterile and ready-to-administer N-acetylcysteine composition according to claim 1; a secondary container enclosing the primary container, and a non-contact oxygen absorbing agent in a space between the primary and secondary container; wherein the primary container contains between 100 and 500 mL of the ready-to-administer N-acetylcysteine composition; and wherein the secondary container is impermeable to light or comprises a metal or metal layer.
 6. The kit of claim 5 wherein the primary container contains 200 mL or 250 mL or 300 mL of the ready-to-administer N-acetylcysteine composition.
 7. The kit of claim 5 wherein the primary and secondary containers are polymeric bags.
 8. The kit of claim 5 wherein the ready-to-administer N-acetylcysteine composition is preservative-free. 